Jeremy L. Marshall

A protein from the salivary glands of the pea aphid, Acyrthosiphon pisum, is essential in feeding on a host plant

In feeding, aphids inject saliva into plant tissues, gaining access to phloem sap and eliciting (and sometimes overcoming) plant responses. We are examining the involvement, in this aphid–plant interaction, of individual aphid proteins and enzymes, as identified in a salivary gland cDNA library. Here, we focus on a salivary protein we have arbitrarily designated Protein C002. We have shown, by using RNAi-based transcript knockdown, that this protein is important in the survival of the pea aphid (Acyrthosiphon pisum) on fava bean, a host plant. Here, we further characterize the protein, its transcript, and its gene, and we study the feeding process of knockdown aphids. The encoded protein fails to match any protein outside of the family Aphididae. By using in situ hybridization and immunohistochemistry, the transcript and the protein were localized to a subset of secretory cells in principal salivary glands. Protein C002, whose sequence contains an N-terminal secretion signal, is injected into the host plant during aphid feeding. By using the electrical penetration graph method on c002-knockdown aphids, we find that the knockdown affects several aspects of foraging and feeding, with the result that the c002-knockdown aphids spend very little time in contact with phloem sap in sieve elements. Thus, we infer that Protein C002 is crucial in the feeding of the pea aphid on fava bean.

Reinforcement: the road not taken

Reinforcement, a process whereby natural selection strengthens prezygotic isolation between sympatric taxa, has gained increasing attention from evolutionary biologists over the past decade. This resurgence of interest is remarkable given that, in the 1980s, most evolutionary biologists considered reinforcement to be, at best, a process that rarely occurred in nature. Although studies of reinforcement are now an important component of speciation research, we still lack a clear understanding of when reinforcement should occur. Theoretical models have suggested that genetic architecture, population structure and the type of selection influence the action of reinforcement. Still to be considered are the consequences of variation in mating system and patterns of sperm or pollen utilization on the likelihood of reinforcement. We argue that traveling down The Road Not Taken (apologies to Frost), that is, taking into consideration mating system and patterns of gamete utilization, leads to novel and more precise predictions of the circumstances under which reinforcement should occur.

Sequence analysis and molecular characterization of larval midgut cDNA transcripts encoding peptidases from the yellow mealworm, Tenebrio molitor L.

Peptidase sequences were analysed in randomly picked clones from cDNA libraries of the anterior or posterior midgut or whole larvae of the yellow mealworm, Tenebrio molitor Linnaeus. Of a total of 1528 sequences, 92 encoded potential peptidases, from which 50 full‐length cDNA sequences were obtained, including serine and cysteine proteinases and metallopeptidases. Serine proteinase transcripts were predominant in the posterior midgut, whereas transcripts encoding cysteine and metallopeptidases were mainly found in the anterior midgut. Alignments with other proteinases indicated that 40% of the serine proteinase sequences were serine proteinase homologues, and the remaining ones were identified as either trypsin, chymotrypsin or other serine proteinases. Cysteine proteinase sequences included cathepsin B‐ and L‐like proteinases, and metallopeptidase transcripts were similar to carboxypeptidase A. Northern blot analysis of representative sequences demonstrated the differential expression profile of selected transcripts across five developmental stages of Te. molitor. These sequences provide insights into peptidases in coleopteran insects as a basis to study the response of coleopteran larvae to external stimuli and to evaluate regulatory features of the response.

Characterization of the multicopper oxidase gene family in Anopheles gambiae

The multicopper oxidase (MCO) family of enzymes includes laccases, which oxidize a broad range of substrates including diphenols, and several oxidases with specific substrates such as iron, copper or ascorbic acid. We have identified five putative MCO genes in the genome of Anopheles gambiae and have cloned cDNAs encompassing the full coding region for each gene. MCO1 mRNA was detected in all developmental stages and in all of the larval and adult tissues tested. We observed an increase in MCO1 transcript abundance in the midguts and Malphighian tubules of adult females following a blood meal and in adult abdominal carcasses in response to an immune challenge. Two alternatively spliced isoforms of MCO2 mRNA were identified. The A isoform of MCO2 was previously detected in larval and pupal cuticle where it probably catalyzes sclerotization reactions (He, N., Botelho, J.M.C., McNall, R.J., Belozerov, V., Dunn, W.A., Mize, T., Orlando, R., Willis, J.H., 2007. Proteomic analysis of cast cuticles from Anopheles gambiae by tandem mass spectrometry. Insect Biochem. Mol. Biol. 37, 135-146). The B isoform was transcriptionally upregulated in ovaries in response to a blood meal. MCO3 mRNA was detected in the adult midgut, Malpighian tubules, and male reproductive tissues; like MCO1, it was upregulated in response to an immune challenge or a blood meal. MCO4 and MCO5 were observed primarily in eggs and in the abdominal carcass of larvae. A phylogenetic analysis of insect MCO genes identified putative orthologs of MCO1 and MCO2 in all of the insect genomes tested, whereas MCO3, MCO4 and MCO5 were found only in the two mosquito species analyzed. MCO2 orthologs have especially high sequence similarity, suggesting that they are under strong purifying selection; the A isoforms are more conserved than the B isoforms. The mosquito specific group shares a common ancestor with MCO2. This initial study of mosquito MCOs suggests that MCO2 may be required for egg development or eggshell tanning in addition to cuticle tanning, while MCO1 and MCO3 may be involved in metal metabolism or immunity.

THE ALLONEMOBIUS-WOLBACHIA HOST-ENDOSYMBIONT SYSTEM: EVIDENCE FOR RAPID SPECIATION AND AGAINST REPRODUCTIVE ISOLATION DRIVEN BY CYTOPLASMIC INCOMPATIBILITY

Abstract Evidence for the evolution of fertilization incompatibilities and rapid speciation can be biased by the occurrence of hybridization and reproductive endosymbionts such as Wolbachia. For example, patterns of mitochondrial DNA (mtDNA) variation can be obscured by mitotypes hitchhiking on extrachromosomal elements like Wolbachia, while such endosymbionts can also induce phenotypes that mirror the operation of intrinsic fertilization incompatibilities between species. Therefore, before strong inferences can be drawn concerning the rates and processes of speciation in arthropod systems, we must first assess whether extrinsic endosymbionts obscure patterns of speciation. Here, I use the Allonemobius fasciatus‐socius species complex to determine what role Wolbachia has played in the presumed rapid divergence of this complex by analyzing patterns of mtDNA and nuclear DNA variation in conjunction with sequence and cytoplasmic incompatibility data on Wolbachia. Data on molecular variation suggest that Wolbachia has not induced a strong selective sweep of the mitochondrial genome; nor does Wolbachia appear to induce cytoplasmic incompatibility. Preliminary evidence indicates that a third species identified within this complex, A. sp. nov. Tex, is partially reproductively isolated from A. socius, its closest relative, via conspecific sperm precedence or some form of postzygotic isolation. Moreover, shared mitotypes between A. sp. nov. Tex and A. socius may indicate the occurrence of a hybrid zone between these species near the border of Texas and Louisiana, although they may also represent shared ancestral polymorphisms. Molecular data also indicate that all three species in this complex diverged from a common ancestor as recently as 3000–30,000 years ago. Finally, the radiation of this complex from its ancestral population likely occurred in the presence of one strain of Wolbachia, thus suggesting a minimal role for Wolbachia during this burst of speciation. In total, barriers to gene flow do appear to have evolved very rapidly in this group of crickets.

Transcriptome Profiling of the Intoxication Response of Tenebrio molitor Larvae to Bacillus thuringiensis Cry3Aa Protoxin

Bacillus thuringiensis (Bt) crystal (Cry) proteins are effective against a select number of insect pests, but improvements are needed to increase efficacy and decrease time to mortality for coleopteran pests. To gain insight into the Bt intoxication process in Coleoptera, we performed RNA-Seq on cDNA generated from the guts of Tenebrio molitor larvae that consumed either a control diet or a diet containing Cry3Aa protoxin. Approximately 134,090 and 124,287 sequence reads from the control and Cry3Aa-treated groups were assembled into 1,318 and 1,140 contigs, respectively. Enrichment analyses indicated that functions associated with mitochondrial respiration, signalling, maintenance of cell structure, membrane integrity, protein recycling/synthesis, and glycosyl hydrolases were significantly increased in Cry3Aa-treated larvae, whereas functions associated with many metabolic processes were reduced, especially glycolysis, tricarboxylic acid cycle, and fatty acid synthesis. Microarray analysis was used to evaluate temporal changes in gene expression after 6, 12 or 24 h of Cry3Aa exposure. Overall, microarray analysis indicated that transcripts related to allergens, chitin-binding proteins, glycosyl hydrolases, and tubulins were induced, and those related to immunity and metabolism were repressed in Cry3Aa-intoxicated larvae. The 24 h microarray data validated most of the RNA-Seq data. Of the three intoxication intervals, larvae demonstrated more differential expression of transcripts after 12 h exposure to Cry3Aa. Gene expression examined by three different methods in control vs. Cry3Aa-treated larvae at the 24 h time point indicated that transcripts encoding proteins with chitin-binding domain 3 were the most differentially expressed in Cry3Aa-intoxicated larvae. Overall, the data suggest that T. molitor larvae mount a complex response to Cry3Aa during the initial 24 h of intoxication. Data from this study represent the largest genetic sequence dataset for T. molitor to date. Furthermore, the methods in this study are useful for comparative analyses in organisms lacking a sequenced genome.

Identification, RNAi knockdown, and functional analysis of an ejaculate protein that mediates a postmating, prezygotic phenotype in a cricket.

Postmating, prezygotic phenotypes, especially those that underlie reproductive isolation between closely related species, have been a central focus of evolutionary biologists over the past two decades. Such phenotypes are thought to evolve rapidly and be nearly ubiquitous among sexually reproducing eukaryotes where females mate with multiple partners. Because these phenotypes represent interplay between the male ejaculate and female reproductive tract, they are fertile ground for reproductive senescence – as ejaculate composition and female physiology typically change over an individuals life span. Although these phenotypes and their resulting dynamics are important, we have little understanding of the proteins that mediate these phenotypes, particularly for species groups where postmating, prezygotic traits are the primary mechanism of reproductive isolation. Here, we utilize proteomics, RNAi, mating experiments, and the Allonemobius socius complex of crickets, whose members are primarily isolated from one another by postmating, prezygotic phenotypes (including the ability of a male to induce a female to lay eggs), to demonstrate that one of the most abundant ejaculate proteins (a male accessory gland-biased protein similar to a trypsin-like serine protease) decreases in abundance over a males reproductive lifetime and mediates the induction of egg-laying in females. These findings represent one of the first studies to identify a protein that plays a role in mediating both a postmating, prezygotic isolation pathway and reproductive senescence.

Comparative Proteomics Uncovers the Signature of Natural Selection Acting on the Ejaculate Proteomes of Two Cricket Species Isolated by Postmating, Prezygotic Phenotypes

Two of the most well-supported patterns to have emerged over the past two decades of research in evolutionary biology are the occurrence of divergent natural selection acting on many male and female reproductive tract proteins and the importance of postmating, prezygotic phenotypes in reproductively isolating closely related species. Although these patterns appear to be common across a wide variety of taxa, the link between them remains poorly documented. Here, we utilize comparative proteomic techniques to determine whether or not there is evidence for natural selection acting on the ejaculate proteomes of two cricket species (Allonemobius fasciatus and A. socius) which are reproductively isolated primarily by postmating, prezygotic phenotypes. In addressing this question, we compare the degree of within-species polymorphism and between-species divergence between the ejaculate and thorax proteomes of these two species. We found that the ejaculate proteomes are both less polymorphic and more divergent than the thorax proteomes. Additionally, we assessed patterns of nucleotide variation for two species-specific ejaculate proteins and found evidence for both reduced levels of variation within species and positive selection driving divergence between species. In contrast, non-species-specific proteins exhibited higher levels of within-species nucleotide variation and no signatures of positive selection. Nucleotide and putative functional data for the two species-specific proteins, along with data for a third protein (ejaculate serine protease), suggest that all three of these genes are candidate speciation genes in need of further study. Overall, these patterns of proteome and nucleotide divergence provide support for the hypothesis that there is a causative link between selection-driven divergence of male ejaculate proteins and the evolution of postmating, prezygotic barriers to gene flow within Allonemobius.

The Genetics of Reproductive Isolation: A Retrospective and Prospective Look with Comments on Ground Crickets

An intriguing aspect of the current renaissance in investigations of the genetics of reproductive isolation is that it has been dominated by studies that resemble work done in the 1930s, 1940s, and 1950s. The dominant model organism (Drosophila), research approaches, and traits of interest (sterility and inviability of hybrids) all harken back to this earlier era. Herein, we explore the factors that led to a rebirth of interest in the genetics of reproductive isolation and to the adoption of the approaches of an earlier generation of biologists. At the same time, we appeal for more intensive investigations of traits that reproductively isolate closely related species, inclusion of a greater range of organisms in studies of reproductive isolation, and focus on a broader range of questions surrounding speciation. We end with a description of ongoing quantitative trait loci (QTL) studies of conspecific sperm precedence in the ground crickets Allonemobius fasciatus and Allonemobius socius. We have found several QTL with large effects on variance in patterns of sperm utilization in backcross females. Moreover, some QTL have an antagonistic effect on conspecific sperm, a finding that lends support to the hypothesis that rapid evolution of conspecific sperm precedence is a by‐product of sexual conflict.

A NEW SPECIES OF BLACK-BELLIED SALAMANDER (GENUS DESMOGNATHUS) FROM THE APPALACHIAN MOUNTAINS OF NORTHERN GEORGIA

We describe a new species of Desmognathus from Union County, Georgia. It closely resembles D. quadramaculatus, but the two are distinguishable by adult size, body proportions, color pattern, and fixed differences at four allozyme loci. The new, smaller species is currently known from two tributaries of the Nottely River and is sympatric with D. quadramaculatus at both sites. The new form is semi-aquatic and utilizes habitats that are similar to those of D. quadramaculatus. Metamorphosis occurs at a smaller size than in D. quadramaculatus, apparently as a result of a shorter larval period. Selection on life history features may have had a role in the origin and divergence of these forms.